SUBSTRATE-SPECIFICITY OF SCHIZOSACCHAROMYCES-POMBE NTH PROTEIN FOR PRODUCTS OF OXIDATIVE DNA-DAMAGE

A gene from Schizosaccharomyces pombe, which encodes a protein with a strong sequence similarity to the Nth protein of Escherichia: coli, ha s recently been identified [Roldan-Arjona, T., Anselmino, C., and Lind ahl, T. (1996) Nucleic Acids Res. 24, 3307-3312]. The functional analy sis of this eukaryotic enzyme indicated that it is a homologue of E. c all Nth protein. The gene has been subcloned and the protein (Nth-Spo) purified to apparent homogeneity. We investigated the substrate speci ficity of this eukaryotic enzyme for modified bases in oxidatively dam aged DNA, using the technique of gas chromatography/isotope-dilution m ass spectrometry (GC/IDMS). DNA substrates containing up to 17 types o f modified bases were prepared by gamma-irradiation or by treatment wi th. H2O2 in the presence of Fe(III)-EDTA or Cu(II). The results reveal ed an efficient excision of five pyrimidine-derived lesions, 5-hydroxy cytosine, thymine glycol, 5-hydroxy-6-hydrothymine: 5,6-dihydroxycytos ine, and 5-hydroxyuracil. None of the other pyrimidine or purine lesio ns was excised. Excision was measured as a function of enzyme concentr ation, time, substrate concentration, and temperature. Kinetic constan ts were determined. Although some DNA base lesions removed by Nth-Spo protein were similar to those previously described for E. coti Nth pro tein, differences between substrate specificities of these two enzymes were noted.